Heterophil-adapted poultry vaccine

ABSTRACT

A method for vaccinating poultry to prevent salmonellosis and other microbial-related health problems in humans is described. The method involves isolation of a poultry heterophil-adapted strain of a microorganism that may be used in a vaccine. A vaccine comprising a preparation of the poultry heterophil-adapted strain is administered to poultry to reduce the transmission of microorganisms causing salmonellosis and other illnesses.

This application is a continuation of PCT Patent Application No.PCT/US96/20555, filed Dec. 18, 1996, now abandoned which claims priorityto U.S. Provisional Patent Application No. 60/009,644, filed Dec. 19,1995.

FIELD OF THE INVENTION

This invention relates to a vaccine for poultry to prevent salmonellosisand other microorganism-related health problems in humans. Inparticular, this invention relates to use of a heterophil-adapted strainof microorganism to vaccinate poultry.

BACKGROUND OF THE INVENTION

Salmonellosis is caused by a group of gram negative bacteria from thegenus Salmonella, and is responsible for approximately 2 million casesof food poisoning annually in the United States. Salmonellosis caused bySalmonella enteritidis (SE) in eggs has been the most importantfood-born public health Salmonella hazard.

Most outbreaks have been traced to consumption of insufficiently cookedeggs. A number of phage types of SE exist but the majority of outbreakshave been traceable to a single or a few phage types of SE. Human SEfood poisoning epidemics have also been reported from many othercountries, but the phage types reported have not necessarily been thoseprevailing in the United States.

Some avian strains of SE are vertically transmitted to the eggs oflaying hens. The ovaries, oviduct and isthmus have been identified assites of vertical transmission. In addition, some observations havepointed to cloacal infection of the egg. It is not known which strainsor phage types of SE are vertically transmitted, and genetic ormolecular requirements for vertical transmission are not known.Infection of hens with SE has led to colonization of the ceca and of thereproductive organs, usually without disease. In most instances infectedhens have continued to lay eggs at normal frequencies. Because ofabsence of overt disease, infection has been difficult to detectclinically or by serologic diagnostic procedures.

Detection of SE in the hen house, individual hens and/or eggs is adifficult task, with a high degree of uncertainty. In the absence oftotal eradication of infection, vaccination remains the method of choicefor disease control. Because SE does not usually cause disease in hens,there is not a vigorous immune response, and hens remain carriers forlong periods, even for life. Therefore, it is presumed that immunizationwill have to be repeatedly administered. Only live attenuated vaccinesgiven orally can be expected to be efficacious in salmonellosis.Curtiss, R. III, et al., Vet. Microbiol. 37: 397-405 (1993). It ispossible that a vaccine strain may have to displace the egg-transmittedstrain in the gut and possibly the reproductive tract of hens.

Salmonellosis due to SE is in most instances not a poultry disease butone of the most serious public health hazards worldwide. Prevention ofthe risk of SE transmission from ingested eggs would save severalthousands of lives and would save around $1 billion annually. Thus, avaccine for poultry against transmission of Salmonella would reduceoccurrence of salmonellosis in humans and make a significantcontribution to public health worldwide.

SUMMARY OF THE INVENTION

The present invention features a method of producing aheterophil-adapted strain of microorganism suitable for use as a live,attenuated vaccine in a poultry species. The method involves incubatingwild-type microorganisms with a first population of heterophils takenfrom a member of a poultry species to generate a sample includingheterophil-internalized microorganisms as well as extracellularmicroorganisms. A substantially pure clone of theheterophil-internalized microorganisms is used for the next step.Microorganisms from the heterophil-internalized clone are incubated witha second population of heterophils to form a next passage ofheterophil-internalized microorganisms and a next passage ofextracellular microorganisms. Preferably the second population ofheterophils is taken from the same member of the poultry species as wasused in the first passage. These procedures are repeated until at leastthree passages, and preferably at least five passages, are completed. Asubstantially pure clone of the heterophil-internalized microorganismsfrom the last passage is isolated to obtain a heterophil-adapted strain.The heterophil-adapted strain can be, although is not necessarily,arginine hydrolase negative.

The poultry species to which this invention is applicable include,without limitation, turkeys, guinea fowl, pigeons, quail, partridge,broiler chickens, and laying hens. The species of microorganism caninclude members of the genus Salmonella, for example Salmonellaenteritidis.

In a preferred embodiment, isolation of a substantially pure clone ofheterophil-internalized microorganism includes incubating theheterophils with an antibiotic, washing, then disrupting the heterophilsto release the internalized microorganisms. The heterophils may beincubated with more than one antibiotic. Antibiotics useful in themethods of the present invention preferably include, without limitation,aminoglycosides, or aminocyclitols. Most preferable is the use ofkanamycin and gentamicin. The antibiotics can be used individually,sequentially or in any combination effective for killing or inactivatingthe relevant extracellular microorganisms.

In another aspect, the invention features a method of vaccinatingpoultry by administering a preparation of a poultry heterophil-adaptedstrain of microorganism to poultry, preferably by an oral route ofadministration. In an alternative embodiment, both a poultryheterophil-adapted strain of microorganism and cholera toxin, a mucosaladjuvant, are provided to the poultry.

In a further aspect, the present invention involves a substantially purepoultry heterophil-adapted strain of microorganism. In preferredembodiments, the microorganism is a member of the genus Salmonella. Mostpreferably, the Salmonella genus member is Salmonella enteritidis. Theheterophil-adapted strain can be arginine hydrolase negative.

In still another aspect, the present invention features a live,attenuated vaccine for poultry comprising a preparation of asubstantially pure poultry heterophil-adapted strain of microorganism.In a preferred embodiment, the microorganism is a member of the genusSalmonella. More preferably, the member of the genus Salmonella isSalmonella enteritidis, for example Salmonella enteritidis strainSETK499, or strain SETK598 having ATCC Accession Number 55770 (TheAmerican Type Culture Collection; 12301 Parklawn Drive, Rockville, Md.20852; Deposited: Apr. 26, 1994). In an alternative embodiment, thevaccine includes a preparation of a substantially pure poultryheterophil-adapted strain of microorganism as well as cholera toxin.Preferably, the vaccine is adapted for oral administration.

In another related aspect, the invention includes a poultry membercomprising a poultry heterophil-adapted strain of microorganism. In apreferred embodiment, the microorganism is a member of the genusSalmonella, for example Salmonella enteritidis. In a further relatedaspect, the invention includes eggs and body parts (wings, breasts,drumsticks, or other body parts sold, for example, for humanconsumption) derived from the immunized poultry member. Such eggs andbody parts are at a lower risk for microorganism contamination than areeggs and body parts derived from non-immunized but otherwise comparablepoultry members.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts fecal shedding of SE wild-type challenge strains SETK474and SETK584 (Experiment SE1).

FIG. 2 depicts fecal shedding of vaccine strain HASES SETK499(Experiment SE1).

FIG. 3 depicts fecal shedding in experiment SE1 of SE wild-typechallenge strains SETK474 and SETK584 before and after exposure tovaccine strain HASES SETK499.

FIG. 4 depicts fecal shedding of vaccine strain HASES SETK598(Experiment SE2).

FIG. 5 depicts fecal shedding in vaccinated hens when challenged withwild-type strain SETK584 post vaccination (Experiment SE2).

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The invention relates to a method of producing a heterophil-adaptedstrain suitable for use as a live, attenuated vaccine for poultry. Suchstrains are useful in reducing the incidence of food-bornmicroorganisms, for example Salmonella enteritidis (SE), causing healthproblems in humans. Specifically, heterophil-adapted strains of SE havebeen isolated and administered orally to laying hens in order toimmunize and prevent egg transmission of SE.

The term heterophil as used herein refers to polymorphonuclear orgranulocytic cells in poultry blood. The term poultry as used hereinrefers to domesticated fowl including without limitation turkeys, guineafowl, pigeons, quail, partridge, broiler chickens and laying hens. Apoultry member as used herein refers to an individual member of apoultry species.

In general, the invention exploits the method of heterophil adaptationfor obtaining vaccines comprising heterophil-adapted strains ofmicroorganisms. In one aspect of the invention, a heterophil-adaptedstrain may be produced by incubating a wild-type strain of amicroorganism with heterophils from a member of a poultry species. Asused herein, the term "wild-type" refers to any microorganism that isnot poultry heterophil-adapted. In a preferred embodiment, the poultryspecies is a laying hen. The microorganism can be, for example, anymember of the genus Salmonella, including without limitation Salmonellaenteritidis (SE), Salmonella gallinarum (SG), Salmonella pullorum (SP),Salmonella dublin (SD), and Salmonella typhimurium (ST). In a preferredembodiment, the microorganism used for heterophil adaptation is SE.Various SE strains may be employed as wild-type microorganisms used forheterophil adaptation, including without limitation wild-type SE strainsSETK474 and SETK584.

Heterophil adaptation may be accomplished in various ways. For example,a wild-type strain of microorganism may be incubated with a firstpopulation of heterophils from a member of a poultry species until someof the microorganisms are internalized by the heterophils. Preferably,the microorganisms are incubated with heterophils for about 30 min. atabout 41° C., although other incubation times may be suitable dependingon incubation temperatures, species of poultry and microorganism, andother conditions. In any case, incubation times are selected to allowfor internalization of at least some microorganisms by the heterophils.Such internalization may be readily monitored by bacteriologic culturesin GN broth and MacConkey agar of disrupted heterophils.

Incubation with heterophils may be performed using whole blood,preferably blood collected in a heparinized tube. Alternatively,purified or partially purified heterophils may be used. In a preferredembodiment, blood from a laying hen is collected in a heparinized tubeand incubated with an overnight culture of the wild-type strain ofmicroorganism. Following overnight incubation, the culture is adjustedto an optical density (O.D.) of about 0.2 at 595 nm in a McFarlandnephelometer. Incubation of poultry heterophils with wild-typemicroorganisms generates a sample containing heterophil-internalizedmicroorganisms as well as microorganisms that are not internalized(i.e., "extracellular" microorganisms) because of a ratio of about 10:1of bacteria:heterophils. Thus, extracellular microorganisms as usedherein refers to microorganisms on the outside surfaces of theheterophils or dispersed in the inoculation medium and unattached to theheterophils. Heterophil-internalized microorganisms as used hereinrefers to microorganisms that have been internalized by the heterophilsduring incubation.

The next step in heterophil adaptation involves isolation of asubstantially pure clone of heterophil-internalized microorganisms. Thisinvolves, first, removal of extracellular microorganisms from theincubation mixture of heterophils and wild-type microorganisms. Anyavailable method of removing extracellular microorganisms may be used.In a preferred embodiment, removal of extracellular microorganismsinvolves incubation with an antibiotic effective for killing orotherwise inactivating the extracellular microorganisms, followed bywashing with media to remove the antibiotic from the sample. Incubationin the presence of an antibiotic should be long enough to kill orinactivate substantially all of the extracellular microorganisms.Preferably, incubation with an antibiotic is for about 60 min at about41° C. The wild-type strain of the microorganism generally should besensitive to the antibiotic used in order to kill or inactivatesubstantially all of the extracellular microorganisms. In a preferredembodiment, samples are incubated with kanamycin or gentamicin in orderto destroy the extracellular microorganisms. In an alternativeembodiment, extracellular microorganisms are killed or inactivated bysequentially incubating with more than one antibiotic. For example, thesample may be first incubated with kanamycin, followed by incubationwith gentamicin. After exposure to antibiotic, the antibiotic isremoved, for example by centrifugation at low speed to pellet theheterophils. The supernatant containing the antibiotic is removed andfresh medium is added to the pellet. The pellet may be resuspended byvortexing.

After killing or inactivating the extracellular microorganisms in thesample and removing any antibiotics, the heterophil-internalizedmicroorganisms may be released by disruption of the washed heterophils.In a preferred embodiment, disruption of the heterophils is performedusing a detergent such as saponin. Then, an aliquot of the disruptedheterophil sample is grown on agar plates or in liquid medium andsubsequently plated on agar plates. In a preferred embodiment, analiquot of about 6 drops of the disrupted heterophil sample isdrop-plated on agar plates. Preferably, the agar plates are MacConkeyagar plates. Following an appropriate period of growth, a singlewell-isolated colony from an agar plate is identified, and represents asubstantially pure clone of heterophil-internalized microorganismscompleting the first passage of heterophil adaptation.

In a second passage, a culture of the cloned heterophil-internalizedmicroorganisms from the first passage is incubated with a secondpopulation of heterophils, preferably from the same source (i.e., thesame bird) as used in the first passage, and is then taken through thesame procedures as described above. Similarly, each subsequent passageis initiated by incubating heterophils from the same bird with a cultureof the cloned heterophil-internalized microorganisms from theimmediately preceding passage.

Heterophil adaptation of a microorganism may be done by starting with awild-type microorganism and performing repeated passages of heterophiladaptation as described above. Preferably, a heterophil-adaptedmicroorganism has been taken through at least three passages ofheterophil adaptation, more preferably through at least four or fivepassages of heterophil adaptation, and even more preferably through atleast six passages of heterophil adaptation. Heterophil adaptation maybe considered successful when the number of colonies recovered after thedetergent disruption of the heterophils diminishes from dozens ofcolonies in the first passage to about 2-5 colonies when plated onMacConkey agar. The diminished number of bacteria surviving inheterophils after each passage may be interpreted as reducedsurvivability in phagocytes.

A heterophil-adapted strain may exhibit a number of genetic orbiochemical changes. For example, loss of arginine hydrolase mayindicate that the heterophil-adapted strain has lost its capability toproduce nitrous oxide (NO), an important oxidative toxic product. It hasbeen learned recently that NO may be the key microbicidal substance inthe killing of Salmonella by macrophages. Thus, in a preferredembodiment, the heterophil-adapted strain is arginine hydrolasenegative. Loss of a virulence plasmid is another example of changesexhibited by some heterophil-adapted strains.

The heterophil-adapted strains can be used for vaccinating poultry. Inorder to vaccinate poultry, a vaccine comprising a preparation of theheterophil-adapted strain is administered. In a preferred embodiment,poultry are vaccinated by oral administration of a preparation ofheterophil-adapted strain of microorganism. Preferably, oraladministration is accomplished by adding a preparation of theheterophil-adapted strain to drinking water. In a preferred embodiment,a preparation of a heterophil adapted strain is added to about 50 mL ofdeionized drinking water. A drinking water cup is left in the cage untilit is substantially empty in order to ensure administration ofsubstantially all of the preparation. The preparation may be in the formof a culture, a frozen sample, a lyophilized sample, an agar stab or anyother form appropriate for maintaining bacteria. In an alternativeembodiment, cholera toxin (a mucosal adjuvant) is provided to thepoultry, in addition to the heterophil-adapted microorganisms. In apreferred embodiment, the preparation contains on the order of about 10⁶to about 10⁸ colony forming units (CFU's) for each bird of theheterophil-adapted strain as administered to the poultry.

The suitability of heterophil-adapted strains as live vaccines forpoultry may be determined in experiments exposing poultry to wild-typechallenge strains and to heterophil-adapted strains. Frequency andlength of shedding, and egg transmissibility, of challenge strains afterexposure to vaccine strains may be used as measures of vaccine efficacy.For example, fecal shedding and egg transmission patterns of vaccine andchallenge strains before and after vaccination may be evaluated andcompared to the same parameters in hens infected with challenge controlstrains.

In one set of experiments, laying hens were exposed to wild-typechallenge strains by addition of the challenge strains to drinkingwater. After two rounds of exposure to challenge strains, the hens wereexposed to a vaccine strain comprising a five-passage (5X)heterophil-adapted strain of SE. The hens were then exposed to challengestrains, then again to the vaccine strain. This set of experimentsestablished that infection with wild-type challenge strains led toprolonged fecal shedding and egg transmission and that there was noconvalescent immunity against fecal excretion and only partialconvalescent immunity against egg transmission. The vaccine strain wasshed at low frequency for a short time in feces and there was no eggtransmission of the vaccine strain. Subsequent exposure of thevaccinated hens to wild-type challenge strains or to vaccine strainsresulted in a reduction in frequency and duration of fecal shedding, andelimination of egg transmission.

In a second set of experiments, a six-passage (6X) heterophil-adaptedstrain was used as a vaccine strain. Laying hens were exposed to thevaccine strain at doses of 10⁶ or 10⁸ CFU's per bird followed byexposure to a wild-type challenge strain. A second group was exposed toa vaccine strain along with cholera toxin, a known mucosal adjuvant,followed by exposure to a challenge strain. These experimentsdemonstrated that low dose exposure does not result in fecal shedding,while administration of a high dose of vaccine strain results in somefecal shedding for a limited time (about 10 to 14 days). The use ofcholera toxin reduced the amount of fecal shedding. The fecal sheddingof the challenge strain was significantly lower in vaccinated groupswhen compared to a challenge control group (unvaccinated).Significantly, neither the vaccine strain nor the challenge strain couldbe isolated from eggs of the vaccinated groups. Repeated vaccinationswith the vaccine strains reduced the level of fecal shedding of thechallenge strain in immunized hens. These experiments indicated thatheterophil-adapted strains are safe and effective as vaccines forpoultry.

The invention will be further understood with reference to the followingillustrative embodiments, which are purely exemplary, and should not betaken as limiting the true scope of the present invention as describedin the claims.

EXAMPLE 1 Heterophil Adaptation of Wild-Type Salmonella EnteritidisReagents

Reagents used included GN Hajna broth (GN) from Difco containing sterileHanks or RPMI 640 medium (RPMI) at a pH of about 7.2, heparinized tubesfor drawing blood, kanamycin at 1 mg/mL in Hanks or RPMI, gentamicin at10 ug/mL in Hanks or RPMI, MacConkey agar (Difco) and a sterile solutionof 1% saponin in deionized water.

Methods

On day 1, an isolated colony of the wild-type strain was grown overnightin GN. On day 2, the following steps were performed. Approximately 5 mLof blood were drawn from a chicken into a heparinized tube. To thistube, 4 mL of sterile Hanks and 1 mL of the overnight inoculum (at adensity of approximately 0.2 O.D. in growth medium at 595λ in McFarlandnephelometer) were added. The tube was incubated at 41° C. for 30 minwith gentle stirring or slow rotation. 1 mL of kanamycin solution inHanks was added to the 10 mL of solution in the tube. The tube was thenincubated at 41° C. for 60 min and centrifuged at low speed (200 g) forabout 10 min. 1 mL of the supernatant was inoculated in GN (wash #1) andalso plated on MacConkey agar (plate #1). The residual supernatant wasremoved from the tube and 10 mL of gentamicin in Hanks were used toresuspend the pellet. The tube was then incubated for 30 min andcentrifuged at low speed (200 g) for 10 min. 1 mL of the supernatant wasinoculated in GN (wash #2) and also plated on a MacConkey agar plate(plate #2). The residual supernatant was removed and the pellet wasresuspended in 20 mL of Hanks. The tube was then incubated for 30 minand centrifuged at low speed (200 g) for 10 min. 1 mL of the supernatantwas inoculated in GN (wash #3) and also plated on a MacConkey agar plate(plate #3). The residual supernatant was removed and 2-3 drops of 1%saponin were added to the cell pellet and the tube vortexed. Then, 2 mLof Hanks was added followed by drop-plating 6 drops of the resultingsuspension on a MacConkey agar plate (plate #4). 25 mL of GN were addedto the tube and the tube was incubated overnight at 41° C.

On day 3, the 1st, 2nd and 3rd washes were inspected for sterility. Ifall washes were sterile, a single colony from plate #4 was subculturedin GN and blood agar in order to obtain isolated colonies. On day 4, anisolated colony was selected and inoculated into GN.

The heterophil adaptation protocol of day 2 through day 4 was repeatedusing heparinized blood from the same bird obtained by sequentialbleeding until at least 5 heterophil passages were accomplished withsterile washes. The adaptation thus represents the interaction of singleclone of SE and the phagocytic cells of an individual bird. In all ofthe heterophil passages, special precautions were taken to avoidaccidental isolation of non-phagocytosed SE. The heterophil adaptationis considered successful if the number of SE colonies recovered aftersaponin lysis of heterophils diminishes gradually from dozens ofcolonies after the first passage to about 2-5 colonies in the six dropsof lysed blood plated on MacConkey agar.

EXAMPLE 2 Bacteriologic Cultures of Feces and Eggs

Chicken feces was cultured according to techniques described by Bichleret al., Am. J. Vet. Res. 57:489-495 (1996); Gast R. K., Poultry Science8:1611-1614 (1993). Briefly, feces was cultured overnight in trypticasesoy broth, subcultured in tetrathionate broth with iodine andsulfadiazine for 2 days, subcultured in Rappaport-Vassiliadis medium,and plated on brilliant green agar. Salmonella-suspect colonies wereidentified by the KSU technique (Difco), and by slide agglutinability ofa bacterial suspension in Salmonella D1 grouping serum (Difco). Eggswere cultured according to a modification of the method of Bichler etal., Am. J. Vet. Res. 57: 489-495 (1996). Briefly, the egg shell washwas followed by alcohol sterilization of the egg shell, then byseparately culturing the inner egg shell membrane, the yolk and thewhite.

EXAMPLE 3 Procedures for Laying Hen Experiments

All laying hen experiments described below were carried out with mature(1-2 years old) Leghorn laying hens. The individually caged hens wereselected for high egg production and sampled five times for absence offecal and egg Salmonella infection (using culture methods as set out inExample 2) prior to selection into the experimental pool. Theexperimental SE strains (wild-type and heterophil-adapted SE strain(HASES) vaccine strains) were given in the drinking water. The drinkingwater was placed in cups, with one cup per cage. Feces was sampled fromthe collection trays once daily, and eggs were collected, refrigeratedand cultured for SE (as described in Example 2) within 24 hours ofcollection. Challenge SE strains and HASES vaccine strains were notseparately identified. The next treatment (vaccine or challenge) was notgiven until the feces and eggs of all hens were negative for SE for aminimum of 10 days.

EXAMPLE 4 Safety and Efficacy of HASES SETK499

HASES SETK499 vaccine strain was derived from wild-type field strainSETK474. HASES SETK499 was 5X heterophil-adapted as described in example1.

Experiment SE1 was carried out in five cycles.

Cycle 1: 12 hens were exposed on each of 3 consecutive days toapproximately 1×10⁸ colony forming units (CFU's) of wild-type strainsSETK474 and SETK584 in 50 mL of drinking water to determine frequencyand duration of fecal shedding, and egg transmission of wild-typestrains. 1 mL of 1×10⁸ CFU's/mL was diluted into 50 mL of deionizeddrinking water. Drinking cups were not emptied or replaced until thehens drank all the water (usually 3-4 hours). Feces and eggs werecultured for SE recovery for 39 days.

Cycle 2: Cycle 2 was started 60 days after the beginning of Cycle 1. The12 hens were re-exposed on two consecutive days to approximately 1×10⁸CFU's/50 mL of deionized drinking water of wild-type strains SETK474 andSETK584 (FIG. 1) in order to determine the possible existence ofconvalescent immunity on fecal shedding and egg transmission uponsubsequent infection with the same strains.

Cycle 3: Cycle 3 was started 60 days after the beginning of Cycle 2.These hens were exposed on each of 3 consecutive days to 1×10⁸ CFUs/50mL of drinking water of HASES vaccine strain SETK499 (FIG. 2). Feces andeggs were sampled for 25 days after exposure to determine frequency andduration of fecal shedding, and egg transmission of the vaccine strain.

Cycle 4: Cycle 4 was started 62 days after the beginning of Cycle 3. Thehens were re-exposed on 2 consecutive days to 1×10⁸ CFUs/50 mL ofdrinking water of wild-type strains SETK474 and SETK584 (FIG. 3). Fecesand eggs were sampled for 39 days after exposure to determine frequencyand duration of fecal shedding, and egg transmission of the challengestrain after vaccination (cycle 3) of hens.

Cycle 5: Cycle 5 was started 70 days after the beginning of Cycle 4. Thehens were re-exposed to 1×10⁸ CFUs/50 mL of drinking water of HASESSETK499 (FIG. 2) to determine the effect of anamnestic immunity onfrequency and duration of fecal shedding and egg transmission.

Results and Discussion

The portions of the experiment encompassing cycles 1, 2 and 4established that wild-type strains SETK474 and SETK584 were validchallenge strains for subsequent studies. The experiments establishedthat challenge with these strains led to prolonged (>25 days postinfection) fecal shedding (FIGS. 1 and 3), as well as egg transmission(6.6% to 9.2% of eggs were contaminated; Table 1). By way of comparison,the contamination rate in naturally SE-infected flocks varies from 0.05%to 0.5%. Experiment SE1 also revealed that prior exposure to thesestrains does not lead to convalescent immunity against fecal excretion(FIG. 1 and Table 1). While prior exposure to wild-type strains did notlead to immunity against fecal excretion, it may lead to partialimmunity against egg transmission. Egg transmission in fact decreased insubsequent infection cycles (data not shown).

The procedures performed in cycles 3 and 5 were designed to establishthat the 5X heterophil-adapted strain HASES SETK499 was shed at lowfrequency and for only a short time in feces (FIG. 2). When HASESSETK499 was given at 2×10⁸ CFU on each of 3 subsequent days (cycle 3),fecal shedding occurred on only 3 occasions, on the first (1 hen),fourth (2 hens), and 7th (1 hen) days post-infection. Subsequentexposure, 34 days after cessation of shedding, to HASES SETK499 (5thcycle) resulted in only 1 hen shedding once on day 4 post-infection(FIG. 2). When hens were challenged with wild-type strain after cycle 3vaccination, shedding of challenge strain ceased on day 10post-infection.

The procedures of cycles 3 and 5 were also designed to establish thatthe heterophil-adapted strain is not transmitted to the egg. NeitherHASES SETK499, nor subsequently administered challenge strain SETK584,were transmitted to the eggs of these hens (Table 1).

In summary, experiment SE1 revealed that HASES SETK499 is a safe andeffective vaccine strain. The strain is shed in feces of infected hensat low frequency for only a short time after oral administration (7days; cycle 3). Shedding was further reduced in frequency and durationupon 2nd infection with this strain (cycle 5), suggesting an anamnesticeffect. Primary vaccination (cycle 3) appeared to have eliminated thechallenge strain infection on day 10 post-infection. (Cycle 4). HASESSETK499 was not transmitted to eggs and appeared to prevent subsequentegg transmission of the challenge strain SETK584.

EXAMPLE 5 Characterization and Efficacy of HASES SETK598

HASES SETK598 vaccine strain was derived from wild-type field strainSETK499 and was 6X heterophil-adapted as described in Example 1.

Experiment SE2 was carried out in two cycles.

Cycle 1 (Vaccine Safety): Ten hens (vaccine group 1) were repeatedlyexposed to HASES SETK598 for primary and anamnestic immunizations. Afteradministration of 3.40×10⁶ CFUs of SETK598 in drinking water, feces andeggs were sampled for 24 days thereafter. The hens were re-exposed 45days after the first exposure to 4.0×10⁶ CFUs and samples were collectedfor 30 days. A third exposure of 5.0×10⁷ CFUs initiated 30 days afterthe second exposure was performed in order to determine the frequencyand duration of fecal shedding and egg transmission. Six hens (vaccinegroup 2) were treated in an identical manner, but were also given 50 ugcholera toxin (CT) in the drinking water on the days of vaccination.Cholera toxin was used because it is a known mucosal adjuvant ofimmunity (FIG. 4). The purpose of cycle 1 was to determine fecalshedding and egg transmission of HASES SETK598 upon repeated vaccinationand to determine any effect of CT on subsequent immunity to challengewith wild-type field strain.

Cycle 2 (vaccine efficacy): Cycle 2 was started 65 days after the end ofCycle 1. The 16 immunized hens (vaccine groups 1 and 2, described above)were exposed to challenge strains. Approximately 1×10⁸ CFUs of wild-typechallenge strain SETK584 were given to these hens. Simultaneousidentical challenge exposure of 11 control hens (FIG. 5) was performedto determine fecal shedding and egg transmission of challenge SE.

Results and Discussion

Experiment SE2 reconfirmed the low and transient intestinal colonizationof HASES, previously shown in experiment SE1 (See Example 4).Vaccination with HASES SETK598 was repeated three times, with 30-day orgreater intervals between vaccinations. Salmonella was not isolatedafter the first and second low-dose vaccination cycles (at approximately3-4×10⁶ CFU per dose; data not shown). When HASES was given at a higherdose in a third immunization cycle (5×10⁷ CFU), it was recovered from 2hens on day 5 post-infection, and from 1 hen on days 6 and 7post-infection from group 1 vaccinated hens. Group 2 hens received 50 ugCT adjuvant with the vaccine, and HASES was recovered once only on day 5post-infection, and once on day 9 post-infection. (FIG. 4). Fecalshedding of the challenge strain was significantly lower in vaccinatedgroups 1 and 2 than in the challenge control group (p<0.01; FIG. 5).Neither HASES nor the challenge SETK584 strain was isolated from any of477 eggs of vaccinated groups 1 and 2, but were isolated from 4 of 71eggs (5.6%) of the challenge control group (Table 1).

Experiment SE2 provided evidence that HASES SETK598 is not shed in fecesof hens given approximately 10⁶ CFU repeatedly. It was shed from fecesof a few hens for up to 9 days post-infection when given at a dose of5×10⁷ CFU. Repeated vaccinations with HASES SETK598 reduced the level offecal shedding of the challenge strain in immunized hens. Neither thevaccine strain nor the challenge strain were found in eggs of vaccinatedhens.

                  TABLE 1                                                         ______________________________________                                        Egg Infection with Wild-Type Strains SETK474 and SETK584                      and Vaccine Strains HASES SETK499 and HASES SETK598                           Wild-type SETK474 and                                                         SETK584             Eggs: No. infected/total                                  ______________________________________                                        First challenge trial                                                                             4/61      6.6%                                            (SE1)                                                                         Second challenge trial                                                                            7/76      9.2%                                            (SE1)                                                                         Challenge trial     4/71      5.6%                                            (SE2)                                                                         Vaccine SE                                                                    5X vaccine HASES SETK499                                                                          0/244     0%                                              (SE1)                                                                         Challenge SE        0/234     0%                                              6X vaccine HASES SETK598                                                                          0/250     0%                                              (SE2)                                                                         Challenge SE        0/227     0%                                              ______________________________________                                    

What is claimed is:
 1. An isolated poultry heterophil-adapted strain ofmicroorganism, wherein said poultry heterophil-adapted strain exhibits agenetic or biochemical change relative to a wild-type strain from whichsaid poultry heterophil-adapted strain is derived.
 2. The strain ofclaim 1, wherein said poultry heterophil-adapted strain is a member ofthe genus Salmonella.
 3. The strain of claim 2, wherein said member isSalmonella enteritidis.
 4. The strain of claim 1, wherein said poultryheterophil-adapted strain lacks a virulence plasmid.
 5. The strain ofclaim 1, wherein said poultry heterophil-adapted strain is argininehydrolase negative.
 6. The strain of claim 1, wherein said poultryheterophil-adapted strain is alive.
 7. The strain of claim 1, whereinsaid poultry heterophil-adapted strain is avirulent to a poultryspecies.
 8. The strain of claim 7, wherein said poultry speciescomprises a species selected from the group consisting of a turkey,guinea, fowl, pigeons, quail, partridge, broiler chicken, and layinghen.
 9. The strain of claim 1, wherein said poultry heterophil-adaptedstrain is in the form of a culture, frozen sample, lyophilized sample,or agar stab.
 10. The strain of claim 1, wherein said poultryheterophil-adapted strain is SETK598 having ATCC Accession Number 55770.11. A vaccine comprising an isolated poultry heterophil-adapted strainof microorganism, wherein administration of said vaccine to a poultryspecies immunizes said poultry species, and wherein said poultryheterophil-adapted strain exhibits a genetic or biochemical changerelative to a wild-type strain from which said poultryheterophil-adapted strain is derived.
 12. The vaccine of claim 11,wherein said poultry heterophil-adapted strain is a member of the genusSalmonella.
 13. The vaccine of claim 11, wherein said member isSalmonella enteritidis.
 14. The vaccine of claim 13, wherein saidadministration reduces or prevents transmission of a Salmonellaenteritidis to an egg of said poultry species.
 15. The vaccine of claim13, wherein said administration reduces or prevents feces shedding of aSalmonella enteritidis by said poultry species.
 16. The vaccine of claim11, wherein said poultry heterophil-adapted strain lacks a virulenceplasmid.
 17. The vaccine of claim 11, wherein said poultryheterophil-adapted strain is arginine hydrolase negative.
 18. Thevaccine of claim 11, wherein said poultry heterophil-adapted strain isalive.
 19. The vaccine of claim 11, wherein said poultryheterophil-adapted strain is avirulent to said poultry species.
 20. Thevaccine of claim 11, wherein said poultry species comprises a speciesselected from the group consisting of a turkey, guinea, fowl, pigeons,quail, partridge, broiler chicken, and laying hen.
 21. The vaccine ofclaim 11, wherein said poultry heterophil-adapted strain is in the formof a culture, frozen sample, lyophilized sample, or agar stab.
 22. Thevaccine of claim 11, wherein said poultry heterophil-adapted strain isSETK598 having ATCC Accession Number
 55770. 23. The vaccine of claim 11,wherein said vaccine is suitable for oral administration.
 24. Thevaccine of claim 11, wherein said vaccine is in aqueous suspension. 25.The vaccine of claim 11, wherein said vaccine is in a unit dosage formand said unit dosage is about 10⁶ to about 10⁸ colony forming units ofsaid poultry heterophil-adapted strain.
 26. The vaccine of claim 11,wherein said vaccine further comprises a mucosal adjuvant.
 27. Thevaccine of claim 26, wherein said mucosal adjuvant comprises choleratoxin.